CRISPR gene editing in pluripotent stem cells reveals the function of MBNL proteins during human in vitro myogenesis.

Alternative splicing has emerged as a fundamental mechanism for the spatiotemporal control of development. A better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of muscleblind-like (MBNL) proteins, a family of tissue-specific splicing regulators whose loss of function is associated with myotonic dystrophy type 1 (DM1), an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the consequences of their losses on the generation of skeletal muscle cells. Our results suggested that MBNL proteins are required for the late myogenic maturation. In addition, loss of MBNL1 and MBNL2 recapitulated the main features of DM1 observed in hiPSC-derived skeletal muscle cells. Comparative transcriptomic analyses also revealed the muscle-related processes regulated by these proteins that are commonly misregulated in DM1. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope with the loss of function of these MBNL proteins.

Cytostatic Effect of Repeated Exposure to Simvastatin: A Mechanism for Chronic Myotoxicity Revealed by the Use of Mesodermal Progenitors Derived from Human Pluripotent Stem Cells.

Statin treatment of hypercholesterolemia can lead to chronic myotoxicity which is, in most cases, alleviated by drug withdrawal. Cellular and molecular mechanisms of this adverse effect have been elusive, in particular because of the lack of in vitro models suitable for long-term exposures. We have taken advantage of the properties of human pluripotent stem cell-derived mesodermal precursors, that can be maintained unaltered in vitro for a long period of time, to develop a model of repeated exposures to simvastatin during more than 2 weeks. This approach unveiled major differences, both in functional and molecular terms, in response to single versus repeated-dose exposures to simvastatin. The main functional effect of the in vitro simvastatin-induced long-term toxicity was a loss of proliferative capacity in the absence of concomitant cell death, revealing that cytostatic effect could be a major contributor to statin-induced myotoxicity. Comparative analysis of molecular modifications induced by simvastatin short-term versus prolonged exposures demonstrated powerful adaptive cell responses, as illustrated by the dramatic decrease in the number of differentially expressed genes, distinct biological pathway enrichments, and distinct patterns of nutrient transporters expressed at the cell surface. This study underlines the potential of derivatives of human pluripotent stem cells for developing new approaches in toxicology, in particular for chronic toxicity testing.

Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes. Also addressed is the development of hPSC-based assays for the tissue and cell types of greatest interest in drug toxicology. The chapter draws on a range of expert opinion within the European Commission/Cosmetics Europe-funded alternative testing cluster SEURAT-1 and consensus from international groups delivering this guidance such as the International Stem Cell Banking Initiative. Accordingly, the chapter summarizes the most up-date best practices in the use and quality control of human Pluripotent Stem Cell lines in the development of in vitro toxicity assays from leading experts in the field.

The ENKI-2 water-jet system versus Dual Knife for endoscopic submucosal dissection of colorectal lesions: a randomized comparative animal study.

BACKGROUND AND STUDY AIMS: The ENKI-2 water-jet system for endoscopic submucosal dissection (ESD) combines submucosal saline pressure injection with dissection. The aim of this study was to compare ENKI-2 with a standard device in terms of procedure time and perforation rate during colorectal ESD. METHODS: In this randomized comparative study, 10 30-mm-diameter lesions were created in the colon and rectum of 10 healthy adult pigs. The ESD procedure time and perforation rates were recorded for the ENKI-2 system and a standard Dual Knife method. Each pig had half the lesions dissected by ENKI-2 and half dissected by Dual Knife. One experienced and one inexperienced endoscopist took part in the study. RESULTS: A total of 95 lesions were dissected (47 by ENKI-2 and 48 by Dual Knife). The experienced endoscopist was able to excise comparably sized 30-mm lesions using both techniques. The dissection time was shorter for ENKI-2 (18.9 vs. 25.6 minutes; P = 0.034) and the perforation rate was lower compared with the Dual Knife (one perforation [4 %] vs. nine perforations [36 %]; P = 0.011). The inexperienced endoscopist performed significantly larger dissections using the ENKI-2 (934 ± 405 mm2 vs. 673 ±â€Š312 mm2; P = 0.021) despite pre-marking similarly sized artificial lesions. Multivariate analysis demonstrated that for all lesions the dissection time was significantly longer for lesions in the proximal colon (P = 0.001) and the distal colon (P < 0.0001) and shorter for the experienced operator (P < 0.0001). ENKI-2 shortened the dissection time, but not significantly (P = 0.093). CONCLUSIONS: In experienced hands, the ENKI-2 system shortens dissection time and reduces the perforation rate. This effect was not statistically significant for an inexperienced operator. Dissection was faster in the rectum than the colon.

Duchenne muscular dystrophy skeletal muscle cells derived from human induced pluripotent stem cells recapitulate various calcium dysregulation pathways.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in premature death. As a major secondary event, an abnormal elevation of the intracellular calcium concentration in the dystrophin-deficient muscle contributes to disease progression in DMD. In this study, we investigated the specific functional features of induced pluripotent stem cell-derived muscle cells (hiPSC-skMCs) generated from DMD patients to regulate intracellular calcium concentration. As compared to healthy hiPSC-skMCs, DMD hiPSC-skMCs displayed specific spontaneous calcium signatures with high levels of intracellular calcium concentration. Furthermore, stimulations with electrical field or with acetylcholine perfusion induced higher calcium response in DMD hiPSC-skMCs as compared to healthy cells. Finally, Mn2+ quenching experiments demonstrated high levels of constitutive calcium entries in DMD hiPSC-skMCs as compared to healthy cells. Our findings converge on the fact that DMD hiPSC-skMCs display intracellular calcium dysregulation as demonstrated in several other models. Observed calcium disorders associated with RNAseq analysis on these DMD cells highlighted some mechanisms, such as spontaneous and activated sarcoplasmic reticulum (SR) releases or constitutive calcium entries, known to be disturbed in other dystrophin-deficient models. However, store operated calcium entries (SOCEs) were not found to be dysregulated in our DMD hiPSC-skMCs model. These results suggest that all the mechanisms of calcium impairment observed in other animal models may not be as pronounced in humans and could point to a preference for certain mechanisms that could correspond to major molecular targets for DMD therapies.

Dystrophin deficiency impairs cell junction formation during embryonic myogenesis.

Mutations in the DMD gene lead to Duchenne muscular dystrophy, a severe X-linked neuromuscular disorder that manifests itself as young boys acquire motor functions. DMD is typically diagnosed at 2 to 4 years of age, but the absence of dystrophin negatively impacts muscle structure and function before overt symptoms appear in patients, which poses a serious challenge in the optimization of standards of care. In this report, we investigated the early consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Here, dystrophin deficiency was linked to marked dysregulations of cell junction protein families involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.

Dystrophin deficiency impairs cell junction formation during embryonic myogenesis from pluripotent stem cells.

Mutations in the DMD gene lead to Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2-4 years of age, but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients, which poses a serious challenge in current standards of care. Here, we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.

Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts.

Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts-the effector cells of muscle growth and regeneration-are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmdmdx-ßgeo myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.

Intrasphincteric injections of autologous muscular cells in women with refractory stress urinary incontinence: a prospective study.

INTRODUCTION AND HYPOTHESIS: Cell therapy for stress urinary incontinence (SUI) management has been experienced with encouraging results. METHODS: We conducted an open prospective study on 12 women presenting severe SUI with fixed urethra, after previous failed surgical management. Patients underwent intrasphincteric injections of autologous progenitor muscular cells isolated from a biopsy of deltoid muscle. Primary endpoint focused on safety (measurement of Q(max) variation after 3 months). Secondary endpoints assessed side effects and efficacy. RESULTS: No variation was diagnosed on Q(max) measurements. Efficacy data show that three of 12 patients are dry at 12 months, seven other patients are improved on pad test but not on voiding diary, and two patients were slightly worsened by the procedure. Quality of life was improved in half of patients. CONCLUSIONS: Cell therapy for severe multioperated cases of SUI is a mini-invasive, feasible, and safe procedure that can improve urinary condition in as a second line therapy.

Myogenesis modelled by human pluripotent stem cells: a multi-omic study of Duchenne myopathy early onset.

BACKGROUND: Duchenne muscular dystrophy (DMD) causes severe disability of children and death of young men, with an incidence of approximately 1/5000 male births. Symptoms appear in early childhood, with a diagnosis made mostly around 4 years old, a time where the amount of muscle damage is already significant, preventing early therapeutic interventions that could be more efficient at halting disease progression. In the meantime, the precise moment at which disease phenotypes arise-even asymptomatically-is still unknown. Thus, there is a critical need to better define DMD onset as well as its first manifestations, which could help identify early disease biomarkers and novel therapeutic targets. METHODS: We have used both human tissue-derived myoblasts and human induced pluripotent stem cells (hiPSCs) from DMD patients to model skeletal myogenesis and compared their differentiation dynamics with that of healthy control cells by a comprehensive multi-omic analysis at seven time points. Results were strengthened with the analysis of isogenic CRISPR-edited human embryonic stem cells and through comparisons against published transcriptomic and proteomic datasets from human DMD muscles. The study was completed with DMD knockdown/rescue experiments in hiPSC-derived skeletal muscle progenitor cells and adenosine triphosphate measurement in hiPSC-derived myotubes. RESULTS: Transcriptome and miRnome comparisons combined with protein analyses demonstrated that hiPSC differentiation (i) leads to embryonic/foetal myotubes that mimic described DMD phenotypes at the differentiation endpoint and (ii) homogeneously and robustly recapitulates key developmental steps-mesoderm, somite, and skeletal muscle. Starting at the somite stage, DMD dysregulations concerned almost 10% of the transcriptome. These include mitochondrial genes whose dysregulations escalate during differentiation. We also describe fibrosis as an intrinsic feature of DMD skeletal muscle cells that begins early during myogenesis. All the omics data are available online for exploration through a graphical interface at https://muscle-dmd.omics.ovh/. CONCLUSIONS: Our data argue for an early developmental manifestation of DMD whose onset is triggered before the entry into the skeletal muscle compartment, data leading to a necessary reconsideration of dystrophin roles during muscle development. This hiPSC model of skeletal muscle differentiation offers the possibility to explore these functions as well as find earlier DMD biomarkers and therapeutic targets.

Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins.

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for "all-in-one" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

The paracrine effects of human induced pluripotent stem cells promote bone-like structures via the upregulation of BMP expression in a mouse ectopic model.

Use of human induced pluripotent stem cells (h-iPSCs) for bone tissue engineering is most appealing, because h-iPSCs are an inexhaustible source of osteocompetent cells. The present study investigated the contribution of undifferentiated h-iPSCs and elucidated aspects of the underlying mechanism(s) of the involvement of these cells to new bone formation. Implantation of undifferentiated h-iPSCs seeded on coral particles in ectopic sites of mice resulted in expression of osteocalcin and DMP-1, and in mineral content similar to that of the murine bone. The number of the implanted h-iPSCs decreased with time and disappeared by 30 days post-implantation. In contrast, expression of the murine osteogenic genes at day 15 and 30 post-implantation provided, for the first time, evidence that the implanted h-iPSCs affected the observed outcomes via paracrine mechanisms. Supporting evidence was provided because supernatant conditioned media from h-iPSCs (h-iPSC CM), promoted the osteogenic differentiation of human mesenchymal stem cells (h-MSCs) in vitro. Specifically, h-iPSC CM induced upregulation of the BMP-2, BMP-4 and BMP-6 genes, and promoted mineralization of the extracellular matrix. Given the current interest in the use of h-iPSCs for regenerative medicine applications, our study contributes new insights into aspects of the mechanism underlying the bone promoting capability of h-iPSCs.

Noncultured, autologous, skeletal muscle cells can successfully engraft into ovine myocardium.

BACKGROUND: There is compelling evidence showing that cellular cardiomyoplasty can improve cardiac function. Considering the potential benefit of using noncultured muscle cells (little time, lower cost, reduced risk of contamination), we investigated the feasibility of grafting cells obtained directly after enzymatic dissociation of skeletal muscle biopsies into ovine myocardium. We hypothesized that those noncultured muscle cells would engraft massively. METHODS AND RESULTS: Autologous, intramyocardial skeletal muscle cell implantation was performed in 8 sheep. A skeletal muscle biopsy sample ( approximately 10 g) was explanted from each animal. The sheep were left to recover for approximately 3 hours and reanesthetized when the cells were ready for implantation. A left fifth intercostal thoracotomy was performed, and 10 epicardial injections of the muscle preparation (between 10 and 20 million cells) were carried out. All sheep were euthanized 3 weeks after myocardial implantation. Immunohistochemistry was performed with monoclonal antibodies to a fast skeletal isoform of myosin heavy chain. Skeletal myosin heavy-chain expression was detected in all slides at 3 weeks after implantation in 8 of 8 animals, confirming engraftment of skeletal muscle cells. Massive areas of engraftment (from 2 to 9 mm in diameter) or discrete loci were noted within the myocardial wall. CONCLUSIONS: Our results indicate that noncultured skeletal muscle cells can successfully and massively engraft in ovine myocardium. Thus, avoiding the cell culture expansion phase is feasible and could become a promising option for cellular cardiomyoplasty.

Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5.

BACKGROUND: The two myogenic regulatory factors Myf5 and MyoD are basic helix-loop-helix muscle transcription factors undergoing differential cell cycle dependent proteolysis in proliferating myoblasts. This regulated degradation results in the striking expression of these two factors at distinct phases of the cell cycle, and suggests that their precise and alternated disappearance is an important feature of myoblasts, maybe connected to the maintenance of the proliferative status and/or commitment to the myogenic lineage of these cells. One way to understand the biological function(s) of the cyclic expression of these proteins is to specifically alter their degradation, and to analyze the effects of their stabilization on cells. To this aim, we undertook the biochemical analysis of the mechanisms governing Myf5 mitotic degradation, using heterologous systems. RESULTS: We show here that mitotic degradation of Myf5 is conserved in non-myogenic cells, and is thus strictly under the control of the cell cycle apparatus. Using Xenopus egg extracts as an in vitro system to dissect the main steps of Myf5 mitotic proteolysis, we show that (1) Myf5 stability is regulated by a complex interplay of phosphorylation/dephosphorylation, probably involving various kinases and phosphatases, (2) Myf5 is ubiquitylated in mitotic extracts, and this is a prerequisite to its degradation by the proteasome and (3) at least in the Xenopus system, the E3 responsible for its mitotic degradation is not the APC/C (the major E3 during mitosis). CONCLUSION: Altogether, our data strongly suggest that the mitotic degradation of Myf5 by the ubiquitin-proteasome system is precisely controlled by multiple phosphorylation of the protein, and that the APC/C is not involved in this process.

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address. METHODS: The bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts. RESULTS: Unexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named "Dp412e" has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model. CONCLUSIONS: In this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.

Dose effect relationship between the number of normal progenitor muscle cells grafted in mdx mouse skeletal striated muscle and the number of dystrophin-positive fibres.

The transplantation of progenitor muscle cells in striated skeletal muscle of mdx mice, a model of dystrophin deficiency, is well known to induce the formation of mosaic fibres expressing dystrophin near the site of injection. We tried to determine if the number of injected cells is related to the number of dystrophin-positive fibres. The grafted cells provided by 5 day-old C57Bl10 mice are syngenic to mdx mice and were cultured to select undifferentiated progenitors. Dystrophin-positive fibres distinct to 'revertant' fibres were detectable 10 days following the graft of as few as 10(3) cells. The number of dystrophin-positive fibres increases logarithmically with the number of grafted cells. The data indicate that the number of dystrophin-positive fibres plateaus above 5x10(5)-10(6) grafted cells and that a greater number of progenitor cells is not required to obtain a better result.

H19 antisense RNA can up-regulate Igf2 transcription by activation of a novel promoter in mouse myoblasts.

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.

Fate of implanted syngenic muscle precursor cells in striated urethral sphincter of female rats: perspectives for treatment of urinary incontinence.

OBJECTIVES: To analyze the outcome of syngenic skeletal muscle precursor cells (MPCs) after implantation in the striated urethral sphincter of the female rat. METHODS: MPCs were isolated from the striated muscles of the lower limbs and infected with a retrovirus carrying the gene for green fluorescent protein. Approximatively 10(5) cells were injected longitudinally in the striated urethral sphincter of 24 animals using a 10-muL Hamilton syringe. The whole urethra was excised at 0, 1, 7, 10, 14, 30, and 90 days after implantation for histologic study and fluorescence analysis of the transections. RESULTS: At days 0 and 1, some small, round, fluorescent MPCs were observed at the injection site. At day 7, significant MPC persistence was noted, with infiltration of inflammatory cells in the whole urethral wall (striated muscle layer, smooth muscle layer, and connective tissue). At day 10, some fusiform cells appeared in the striated muscle layer, suggesting the incorporation of MPCs into the striated myofibers. Inflammatory cells were no longer visible. At day 14, the fusiform cells tended to be larger. The small, round cells were no longer seen. At days 30 and 90, all myofibers of the striated muscle layer were strongly fluorescent, and no fluorescence was detectable in the smooth muscle layer. CONCLUSIONS: Implantation of skeletal MPCs in the urethral sphincter resulted in selective incorporation into striated myofibers. Muscle-derived cell autografting could represent a new approach for the treatment of urinary incontinence in humans.